Evidence for a Conformational Change in Actin Induced by Fimbrin (N375) Binding

Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

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Disruption of the utrophin–actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

Biochemical Journal ◽

10.1042/bj3370119 ◽

1998 ◽

Vol 337 (1) ◽

pp. 119-123 ◽

Cited By ~ 2

Author(s):

Glenn E. MORRIS ◽

Nguyen thi MAN ◽

Nguyen thi Ngoc HUYEN ◽

Alexander PEREBOEV ◽

John KENDRICK-JONES ◽

...

Keyword(s):

Binding Sites ◽

Three Dimensional ◽

Binding Domain ◽

Actin Binding ◽

Dimensional Structure ◽

Functional Regions ◽

Binding Surface ◽

Actin Binding Domain ◽

Actin Binding Site ◽

Helical Region

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin–F-actin binding and was shown to recognize a predicted helical region at residues 13–22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.

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Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

The Journal of Cell Biology ◽

10.1083/jcb.126.2.433 ◽

1994 ◽

Vol 126 (2) ◽

pp. 433-443 ◽

Cited By ~ 124

Author(s):

A McGough ◽

M Way ◽

D DeRosier

Keyword(s):

Image Analysis ◽

Smooth Muscle ◽

Binding Sites ◽

Actin Filaments ◽

Three Dimensional ◽

Actin Binding ◽

Dimensional Structure ◽

Outer Face ◽

Actin Binding Domain

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

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Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

10.1101/2020.04.14.041046 ◽

2020 ◽

Author(s):

Yuuki Hayakawa ◽

Masak Takaine ◽

Taiga Imai ◽

Masafumi D. Yamada ◽

Keiko Hirose ◽

...

Keyword(s):

Fission Yeast ◽

Actin Filaments ◽

Structural Changes ◽

Myosin Ii ◽

Binding Domain ◽

Actin Binding ◽

Skeletal Muscle Myosin ◽

Actin Binding Domain ◽

Muscle Myosin

AbstractThe contraction of contractile rings (CRs) depends on interaction between actin filaments and myosin II filaments. The rate of contraction in the fission yeast Schizosaccharomyces pombe is less than 1/120 of the velocity of acto-myosin II movement in vitro, but the mechanism of inhibition has not been described. Here, we found that the calponin-homology actin binding domain of fission yeast IQGAP Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on skeletal muscle myosin II fragments in vitro, even at a low ratio of bound Rng2CHD to actin protomers, reducing the sliding velocity to half when the binding ratio was 1/75. Rng2CHD also induced structural changes of actin filaments and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II carrying ADP. Intriguingly, actin-activated ATPase of S1 was only mildly inhibited, even by high concentrations of Rng2CHD. Moreover, the motility of actin filaments by myosin V was not inhibited by Rng2CHD. We propose a new regulatory mechanism for acto-myosin II movement that involves Rng2CHD-induced structural changes of actin filaments.

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αE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors

Molecular Biology of the Cell ◽

10.1091/mbc.e13-07-0388 ◽

2013 ◽

Vol 24 (23) ◽

pp. 3710-3720 ◽

Cited By ~ 62

Author(s):

Scott D. Hansen ◽

Adam V. Kwiatkowski ◽

Chung-Yueh Ouyang ◽

HongJun Liu ◽

Sabine Pokutta ◽

...

Keyword(s):

Actin Filament ◽

Actin Filaments ◽

Binding Domain ◽

Actin Binding ◽

Filamentous Actin ◽

Filament Structure ◽

Actin Binding Domain ◽

Filament Bundles ◽

Underlying Mechanisms ◽

Cell Cell

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell–cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments and that binding is accompanied by a conformational change in the actin protomer that affects filament structure. αE-catenin ABD binding limits barbed-end growth, especially in actin filament bundles. αE-catenin ABD inhibits actin filament branching by the Arp2/3 complex and severing by cofilin, both of which contact regions of the actin protomer that are structurally altered by αE-catenin ABD binding. In epithelial cells, there is little correlation between the distribution of αE-catenin and the Arp2/3 complex at developing cell–cell contacts. Our results indicate that αE-catenin binding to filamentous actin favors assembly of unbranched filament bundles that are protected from severing over more dynamic, branched filament arrays.

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Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

The Journal of Cell Biology ◽

10.1083/jcb.96.5.1400 ◽

1983 ◽

Vol 96 (5) ◽

pp. 1400-1413 ◽

Cited By ~ 71

Author(s):

R Niederman ◽

P C Amrein ◽

J Hartwig

Keyword(s):

Actin Filaments ◽

Binding Protein ◽

Three Dimensional ◽

Actin Binding ◽

Dimensional Structure ◽

Molar Ratio ◽

Computer Assisted ◽

Three Dimensional Structure ◽

Actin Binding Protein ◽

Critical Point Drying

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

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Z-disc-associated, Alternatively Spliced, PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy

Journal of Biological Chemistry ◽

10.1074/jbc.m114.550418 ◽

2014 ◽

Vol 289 (19) ◽

pp. 13615-13626 ◽

Cited By ~ 27

Author(s):

Xiaoyan Lin ◽

Janelle Ruiz ◽

Ilda Bajraktari ◽

Rachel Ohman ◽

Soojay Banerjee ◽

...

Keyword(s):

Skeletal Muscle ◽

Actin Filaments ◽

Binding Domain ◽

Actin Binding ◽

Muscle Actin ◽

Myofibrillar Myopathy ◽

Actin Binding Domain ◽

Alternatively Spliced

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Focal segmental glomerulosclerosis ACTN4 mutants binding to actin: regulation by phosphomimetic mutations

Scientific Reports ◽

10.1038/s41598-019-51825-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Hanshuang Shao ◽

Bentley Wingert ◽

Astrid Weins ◽

Martin R. Pollak ◽

Carlos Camacho ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Focal Segmental Glomerulosclerosis ◽

Actin Filaments ◽

Binding Activity ◽

Binding Domain ◽

Actin Binding ◽

Dominant Form ◽

Cell Functions ◽

Segmental Glomerulosclerosis ◽

Actin Binding Domain

Abstract Natural mutations such as lysine 255 to glutamic acid (K to E), threonine 259 to isoleucine (T to I) and serine 262 to proline (S to P) that occur within the actin binding domain of alpha-actinin-4 (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS) in affected humans. This appears due to elevated actin binding propensity in podocytes resulting in a ‘frozen’ cytoskeleton. What is challenging is how this cellular behavior would be compatible with other cell functions that rely on cytoskeleton plasticity. Our previous finding revealed that wild type ACTN4 can be phosphorylated at tyrosine 4 and 31 upon stimulation by epidermal growth factor (EGF) to reduce the binding to actin cytoskeleton. We queried whether the elevated actin binding activity of FSGS mutants can be downregulated by EGF-mediated phosphorylation, to discern a mechanism by which the actin-cytoskeleton can be released in FSGS. In this manuscript, we first constructed variants with Y4/31E to mimic the phosphorylation at tyrosines 4 and 31 based on earlier modeling simulations that predicted that this would bury the actin binding domains and lead to a decrease in actin binding activity. We found that Y4/31E significantly reduced the actin binding activity of K255E, T259I and S262P, dramatically preventing them from aggregating in, and inhibiting motility of, podocytes, fibroblasts and melanoma cells. A putative kinase target site at Y265 in the actin binding domain was also generated as a phosphomimetic ACTN4 Y265E that demonstrated even greater binding to actin filaments than K255E and the other FSGS mutants. That the tyrosine kinase regulation of FSGS mutation binding to actin filaments can occur in cells was shown by phosphorylation on Y4 and Y31 of the K225E after extended exposure of cells to EGF, with a decrease in ACTN4 aggregates in fibroblasts. These findings will provide evidence for targeting the N-termini of FSGS ACTN4 mutants to downregulate their actin binding activities for ameliorating the glomerulosclerotic phenotype of patients.

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Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin.

The Journal of Cell Biology ◽

10.1083/jcb.126.5.1231 ◽

1994 ◽

Vol 126 (5) ◽

pp. 1231-1240 ◽

Cited By ~ 102

Author(s):

A R Menkel ◽

M Kroemker ◽

P Bubeck ◽

M Ronsiek ◽

G Nikolai ◽

...

Keyword(s):

Actin Filaments ◽

Direct Interaction ◽

Binding Domain ◽

Actin Binding ◽

Filamentous Actin ◽

Permeabilized Cells ◽

Cell Matrix ◽

Actin Binding Domain ◽

Major Structural Component

Vinculin, a major structural component of vertebrate cell-cell and cell-matrix adherens junctions, has been found to interact with several other junctional components. In this report, we have identified and characterized a binding site for filamentous actin. These results included studies with gizzard vinculin, its proteolytic head and tail fragments, and recombinant proteins containing various gizzard vinculin sequences fused to the maltose binding protein (MBP) of Escherichia coli. In cosedimentation assays, only the vinculin tail sequence mediated a direct interaction with actin filaments. The binding was saturable, with a dissociation constant value in the micromolar range. Experiments with deletion clones localized the actin-binding domain to a region confined by residues 893-1016 in the 170-residue-long carboxyterminal segment, while the proline-rich hinge connecting the globular head to the rodlike tail was not required for this interaction. In fixed and permeabilized cells (cell models), as well as after microinjection, proteins containing the actin-binding domain specifically decorated stress fibers and the cortical network of fibroblasts and epithelial cells, as well as of brush border type microvilli. These results corroborated the sedimentation experiments. Our data support and extend previous work showing that vinculin binds directly to actin filaments. They are consistent with a model suggesting that in adhesive cells, the NH2-terminal head piece of vinculin directs this molecule to the focal contact sites, while its tail segment causes bundling of the actin filament ends into the characteristic spear tip-shaped structures.

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